1. Field of the Invention
This invention relates to the field of commercial biocatalytic production of chemical intermediates useful in the manufacture of high quality engineering thermoplastics. It provides a fermentation process for the production of .alpha.,.omega.-alkanedicarboxylic acids by yeast using readily available raw materials which achieves a high specific productivity. More specifically, the invention provides fermentation conditions which yield optimal performance of the yeast biocatalyst in the conversion of fatty acids, fatty acid esters, or alkanes to .alpha.,.omega.-alkanedicarboxylic acids.
2. Description of the Prior Art
Long-chain .alpha.,.omega.-alkanedicarboxylic acids with a carbon number of nine or greater (hereinafter referred to as diacids) are used as intermediates in the synthesis of a wide variety of chemical products, particularly in the production of plastics and other bulk specialty chemicals. In particular, these diacids are used in the production of a copolyestercarbonate (LEXAN.TM.) which retains high impact strength, the hallmark of polycarbonate resin, while offering superior melt and flow characteristics relative to standard (bisphenol-A; BPA) polycarbonate. These properties make plastics which are useful for applications requiring light, thin-walled yet strong parts.
Diacids are currently produced almost exclusively by non-biological conversion processes, based upon the use of nonrenewable petrochemical feedstock. These multi-step chemical conversion processes typically produce unwanted hazardous byproducts which both result in yield losses and must be destroyed before they are released to the environment. Disposal of a hazardous waste stream greatly adds to the cost of production. In addition, the organic chemical synthesis of long-chain diacids is limited by the starting materials used, and each chemical synthesis process can produce only one species of diacid.
For example, according to prior art methods, dodecanedioic acid is produced by a multi-step chemical conversion process that has significant limitations and disadvantages. Because the synthetic process begins with the starting material butadiene (a 4-carbon petrochemical), the only diacids which can be synthesized are those with a carbon number which is a multiple of four. In practice, only dodecanedioic acid is made by this process, and dodecanedioic acid is the longest straight chain diacid currently available using an industrial chemical synthesis process. The dodecanedioic acid process produces byproducts such as cyclooctadiene and vinyl cyclohexene, which result in yield losses, and nitrogen oxides, which are either released to the atmosphere or must be destroyed in a reduction furnace.
Production of diacids using bioconversion is a potentially promising method which may overcome some of the disadvantages of the current chemical processes. Among the advantages inherent in all biological conversion processes are the ability to use renewable resources as starting materials for the process rather than petrochemicals, and the ability to produce chemicals without also producing a hazardous waste stream, disposal of which is expensive. For example, diacids may be produced from inexpensive long-chain fatty acids, which are readily available from renewable agricultural and forest products such as soybean oil, tallow, corn oil, and tall oil, without the production of the dangerous waste products discussed above.
Diacids are produced in only a single step when a biological process is used. Moreover, a bioconversion process can be adapted easily to produce a wide range of diacids, since the biocatalyst accepts a variety of starting materials. Therefore, a bioconversion method can produce diacids of different lengths which were unavailable for practical reasons using prior art chemical methods.
Of particular importance, a biocatalyst can produce diacids with longer chain lengths. Diacids with a carbon number of 16 or 18 could be produced using the same basic bioconversion which produces other diacids. These longer chain diacids are effective at lowering of melt viscosity in the copolyestercarbonate at a lower diacid concentration than the C12 diacid, and are thus more economical to use. Using prior art methods, however, these longer chain diacids can not be produced commercially and are currently unavailable for widespread use.
More importantly from a business perspective, however, biological conversion processes have the potential to produce diacids for a lower cost than the currently available chemical process. To do this, any biotechnological process must be able to utilize inexpensive, easily available organic substrates as starting materials, and convert those substrates to the desired diacid product with high efficiency.
The biological conversion process for production of long chain aliphatic diacids is carried out by batch fermentation. The batch fermentation process consists of two phases: growth and conversion (or transformation). The growth phase is initiated by inoculating a batch fermenter containing a nutrient medium with the yeast biocatalyst. During this phase of the process, the cells increase in number to a cell density which is dependent on many factors, including the cell type and the nutrient content of the medium. Growth continues in the batch fermenter under selected conditions for a selected period of time or until a selected cell density is reached, at which time the fatty acid, fatty acid ester, or alkane substrate is added to initiate the conversion phase, during which the desired product is formed. During conversion, an excess of substrate is always maintained. A carbon source (cosubstrate) such as glucose also is added throughout the conversion phase to provide an energy source for the yeast. When conversion is completed, the yeast biomass is separated from the fermentation medium, and the diacid product is recovered and purified from the solution.
Yeast produce diacids from fatty acids through the .omega.-oxidation pathway. The first and rate-limiting step is the oxidation of the terminal methyl carbon to produce an .omega.-hydroxy acid. This step is mediated by a membrane- bound enzyme complex consisting of a cytochrome P450 monooxygenase and an associated NADPH cytochrome reductase. Two additional enzymes, an alcohol oxidase and an aldehyde dehydrogenase, further oxidize the alcohol to create an .omega.-aldehyde acid and then the corresponding .alpha.,.omega.-dicarboxylic acid. Several yeasts are known to produce various diacids when grown on fatty acid, fatty acid ester, or alkane substrates, for example Candida tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis, and C. zeylenoides.
These yeasts have a number of limitations which prevent their efficient use for the commercial production of diacids, however. In general, the biocatalytic fermentations known heretofore have produced too low a total yield of diacid relative to the fatty acid or alkane starting material to be practically useful. In addition, the yeast biocatalysts produced too large a quantity of unwanted byproducts such as 3-hydroxy acids, and shorter chain acids and diacids due to .beta.-oxidation which reduce the yield and necessitate further steps in the process to remove the impurities. In summary, the prior art biofermentation processes do not produce an efficient conversion of the starting material into the desired diacid product.
The rate of conversion of the starting material to the desired end product (the specific productivity, grams of product formed per liter fermentation medium per hour) also is a key factor in the total effective cost of commercial biological production of diacids. Therefore increasing the specific productivity of the bioconversion process can significantly reduce the diacid production cost.
Recently, genetically modified strains of the yeast Candida tropicalis have been developed in the hope of increasing the specific production of diacids above that obtainable with wild type yeast. U.S. Pat. No. 5,254,466 discloses a genetically modified strain of C. tropicalis (strain H5343, ATCC No. 20962) in which the genes coding for enzymes in the first step of fatty acid .beta.-oxidation have been disrupted so that the yeast can no longer use fatty acids as a carbon source. In this strain, .beta.-oxidative degradation of the substrate and product leading to a progressive shortening of the alkane chains does not occur. This yeast is able to produce highly pure diacids in substantially quantitative yield, without the production of these unwanted byproducts.
U.S. Pat. No. 5,620,878 discloses a C. tropicalis strain termed AR40 (ATCC No. 20987) which has been further modified. Multiple copies of cytochrome P450 and reductase genes of the 4-hydroxylase system have been introduced. This genetic manipulation resulted in a yeast with increased .omega.-hydroxylase activity and even greater specific productivity (g/L/hr) of diacids from long-chain fatty acids. Nevertheless, the preferred process described for genetically modified yeasts in U.S. Pat. No. 5,254,466 (C. tropicalis, strain H5343, ATCC No. 20962); in U.S. Pat. No. 5,620,878; and in a related journal publication, (S. Picataggio, Bio/Technology 10:894-8 (1992)), do not produce the diacids with sufficient specific productivity for their commercial production to be economically feasible.
For biological production of diacids as bulk chemical intermediates to be commercially practical, the cost of production must be significantly reduced. There is consequently a need for a biological fermentation process in which specific productivity is enriched and the efficiency of diacid production is maximized.